Royal Reader Proteins: role in KSHV RNA processing to novel antiviral approaches

KSHV is an oncogenic virus required for the development of Kaposi's sarcoma (KS) and several lymphoproliferative diseases. There are no specific KSHV antivirals or vaccines and current treatments are not targeted, relying on rebuilding the immune system or using cytotoxic agents. As KS is an AIDS-defining disease, controlling HIV/AIDS using immune reconstitution has been investigated as a KS treatment. However, even with effective Antiretroviral Therapy (ART) and well-controlled HIV infection, many patients still develop progressive KS. HIV-KS patients can also exhibit a worsening of symptoms upon ART, developing KS-associated immune reconstitution inflammatory syndrome, now a major contributor to KSHV-associated deaths. Consequently, anti-KSHV therapies are urgently needed.

KSHV has two distinct life cycles, a persistence life-long infection (latency) and an infectious productive cycle (lytic replication). Lytic replication plays an essential role in KSHV pathogenesis. Therefore, it is essential to study the molecular mechanisms which regulate lytic replication, leading to new opportunities for novel antiviral strategies.

We have exciting data showing that KSHV manipulates the host cell N6-methyladenosine (m6A) pathway to promote lytic replication. m6A is the most abundant internal modification of mRNAs. Importantly, m6A exerts its influence over all aspects of RNA metabolism by recruiting effector proteins, m6A readers, which direct the m6A-modified RNA towards distinct biological fates. m6A is now thought to regulate a wide range of physiological process and aberrant m6A is implicated in multiple human diseases. Therefore, KSHV manipulation of m6A presents a novel opportunity to examine how m6A dysregulation impacts gene expression and how this leads to disease. Moreover, it provides an opportunity to develop novel therapeutic approaches.

We highlight that KSHV manipulation of the host m6A pathway has a pro-viral effect. The KSHV transcriptome is heavily m6A-modified and this helps to stabilise specific viral mRNAs by recruiting a new group of m6A reader proteins, known as Royal proteins. These results are reinforced by experiments showing that depletion of the Royal protein, SND1, has a dramatic effect on KSHV ORF50 transcript stability and resulting virus replication. In addition, transcriptome-wide m6A-seq analysis highlights that cellular mRNAs are differentially m6A-modified which strongly correlates with an increase in their expression during infection.

We will now address the following key questions: (i) We will examine what specific features within KSHV mRNAs, apart from m6A modification, enables different Royal proteins to bind preferentially to specific m6A-modified KSHV RNAs. We will use transcriptome-wide analysis to identify the groups of m6A-modified RNAs bound by each Royal protein and characterise any consensus sequences or motifs in these RNAs. Moreover, we will determine if RNA structure enhances Royal protein selectivity. (ii) We will determine why some cellular mRNAs are differentially m6A-modified during the course of infection. It may be the case that these RNAs are required by KSHV to enhance its replication or they are a response by the host cell upon infection. (iii) We have identified RNA-binding small molecules which can preferentially bind to the m6A-modified form of the KSHV ORF50 transcript and reduce KSHV lytic replication. We will fully characterise the activity of these inhibitory small molecules and optimise their function using pharmacophore-based design.

In summary, we will identify novel ways KSHV manipulates the host to enhance its own replication and provide a better understanding of how the dynamic m6A RNA modification regulates gene expression. This will impact on our understanding of the emerging role of m6A in cell and developmental processes, the development of human disease and provide new strategies for therapeutic intervention of an important humuan pathogen.

Widespread distribution of m6A in cellular RNAs has been revealed in a range of eukaryotic species. Conservation of m6A sites across species reinforces a fundamental role for this RNA modification in regulating gene expression and highlights that aberrant m6A regulation is implicated in a range of human diseases.

We have exciting data demonstrating that the oncogenic herpesvirus, KSHV, manipulates the m6A pathway to enhance its own replication. We show that the KSHV transcriptome is heavily m6A-modified and viral m6A-modified RNAs specifically recruit various Royal proteins in a m6A-dependent manner. In addition, we show that during KSHV infection some cellular mRNAs are differentially m6A-modified leading to an increase in their expression. Finally, we have shown that recruitment of the Royal protein, SND1, is essential for KSHV ORF50 RNA stability and depletion of SND1 is detrimental for KSHV lytic replication.

We will now examine the role of specific Royal protein m6A readers in virus replication and determine how they selectively bind specific viral m6A-modified RNAs, examining how RNA sequence and structure enhance Royal protein recruitment. We will also assess the role of prioritised differentially m6A-modified cellular mRNAs during infection. Finally, we will assess and optimise the antiviral activity of RNA-binding small molecules which preferentially bind to the m6A-modified form of the KSHV ORF50 transcript.

This project will determine how KSHV manipulation of the m6A pathway can modulate host and viral gene expression during infection. It will also assess whether disrupting m6A reader recruitment to KSHV m6A-modified RNAs is a viable antiviral approach. Together, this project will provide valuable information on the fundamental role and regulatory aspects of this dynamic RNA modification.